Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jul 12;14(4):R104.
doi: 10.1186/bcr3224.

Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met

Kelly L Mueller, Julie M Madden, Gina L Zoratti, Charlotte Kuperwasser, Karin List, Julie L Boerner

Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met

Kelly L Mueller et al. Breast Cancer Res. .

Abstract

Introduction: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown clinical efficacy in lung, colon, and pancreatic cancers. In lung cancer, resistance to EGFR TKIs correlates with amplification of the hepatocyte growth factor (HGF) receptor tyrosine kinase Met. Breast cancers do not respond to EGFR TKIs, even though EGFR is overexpressed. This intrinsic resistance to EGFR TKIs in breast cancer does not correlate with Met amplification. In several tissue monoculture models of human breast cancer, Met, although expressed, is not phosphorylated, suggesting a requirement for a paracrine-produced ligand. In fact, HGF, the ligand for Met, is not expressed in epithelial cells but is secreted by fibroblasts in the tumor stroma. We have identified a number of breast cancer cell lines that are sensitive to EGFR TKIs. This sensitivity is in conflict with the observed clinical resistance to EGFR TKIs in breast cancers. Here we demonstrate that fibroblast secretion of HGF activates Met and leads to EGFR/Met crosstalk and resistance to EGFR TKIs in triple-negative breast cancer (TNBC).

Methods: The SUM102 and SUM149 TNBC cell lines were used in this study. Recombinant HGF as well as conditioned media from fibroblasts expressing HGF were used as sources for Met activation. Furthermore, we co-cultured HGF-secreting fibroblasts with Met-expressing cancer cells to mimic the paracrine HGF/Met pathway, which is active in the tumor microenvironment. Cell growth, survival, and transformation were measured by cell counting, clonogenic and MTS assays, and soft agar colony formation, respectively. Student's t test was used for all statistical analysis.

Results: Here we demonstrate that treatment of breast cancer cells sensitive to EGFR TKIs with recombinant HGF confers a resistance to EGFR TKIs. Interestingly, knocking down EGFR abrogated HGF-mediated cell survival, suggesting a crosstalk between EGFR and Met. HGF is secreted as a single-chain pro-form, which has to be proteolytically cleaved in order to activate Met. To determine whether the proteases required to activate pro-HGF were present in the breast cancer cells, we utilized a fibroblast cell line expressing pro-HGF (RMF-HGF). Addition of pro-HGF-secreting conditioned fibroblast media to TNBC cells as well as co-culturing of TNBC cells with RMF-HGF fibroblasts resulted in robust phosphorylation of Met and stimulated proliferation in the presence of an EGFR TKI.

Conclusions: Taken together, these data suggest a role for Met in clinical resistance to EGFR TKIs in breast cancer through EGFR/Met crosstalk mediated by tumor-stromal interactions.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Hepatocyte growth factor protects triple-negative breast cancer cells from gefitinib treatments. (A) SUM102 and SUM149 cells were plated at 35,000 cells/well of a six-well plate on day 0. Under normal growth conditions, 50 ng/ml hepatocyte growth factor (HGF) and/or 0.5 μM gefitinib was added to the cells on day 1 and subsequently every other day. Cells were counted on days 1, 4, and 8 using a Coulter Counter. (B) SUM102 and SUM149 cells under normal growth conditions were treated with increasing concentrations of gefitinib in the presence or absence of 50 ng/ml HGF for 7 days. Cells were trypsinized and replated at 4,000 cells/35-mm dish and grown under normal growth conditions for 7 days. Colonies were stained with crystal violet and counted using a cell counter. The percentage of surviving cells was calculated by dividing the number of colonies on the treated plate by the number of colonies on the untreated plate. (C) Under normal growth conditions, SUM102 and SUM149 cells were plated in a layer of agar sandwiched between two layers of agar of different percentage. Every other day for 3 weeks, 50 ng/ml HGF and/or 0.5 μM gefitinib were added to the top layer in a final volume of 300 μl. Colonies were stained with p-iodonitrol and counted using a cell counter. Each experiment was performed in triplicate at least three times. Student's t test was used to determine significance between gefitinib-treated and gefitinib + HGF-treated cells. *P < 0.05. UT, untreated.
Figure 2
Figure 2
Hepatocyte growth factor-mediated cell survival requires epidermal growth factor receptor expression. SUM102 cells were transduced with lentivirus expressing a non-silencing control or four nonoverlapping epidermal growth factor receptor (EGFR) small hairpin RNA (shRNA) targeting sequences. In addition, SUM102 cells were treated with gefitinib (gef) or hepatocyte growth factor (HGF), or were transfected with pcDNA3 or KD-EGFRmt as indicated. (A) Four days after transduction and/or transfection, cells were lysed, and lysates were used to determine expression levels of EGFR via immunoblotting. β-actin was used as a loading control. (B) Duplicate cells were trypsinized and replated in a 96-well plate. One series of gefitinib-treated cells were used as a control to demonstrate HGF protection from gefitinib treatment (far-left bar). Remaining cells were treated with or without HGF at 50 ng/ml without gefitinib for 72 hours. In addition, cells were transfected with KD-EGFRmt at the same time as transduction with shRNA (far-right bar). Cell viability was measured using MTS assays. Numbers under the immunoblot indicate relative densitometry. Lines indicate comparison samples. Each experiment was performed in triplicate at least three independent times. *Gef ± HGF, P < 0.001; -HGF ± EGFR knockdown, P = 0.001. N.S., not significant; NV, no virus; NS, nonsilencing. #1, #2, #3, and #4, arbitrary shRNA clone numbers. KD-EGFR, kinase-dead EGFR; EGFR*, mutated sequence within the shRNA targeting region.
Figure 3
Figure 3
Hepatocyte growth factor stimulates Met phosphorylation and enhances clonogenic survival in the presence of gefitinib. (A) SUM102 and SUM149 cells were cultured with condition media from RMF-HGF fibroblasts for 1 hour in the presence or absence of 0.5 μM gefitinib (gef). Lysates were prepared and analyzed for Met phosphorylation using immunoblotting. Met and β-actin expression levels were used as controls. CM, pro-hepatocyte growth factor (pro-HGF) conditioned media; UT, untreated. (B) SUM102 and SUM149 cells were treated with conditioned media from RMF-HGF fibroblasts in the presence or absence of the indicated concentration of gefitinib or erlotinib for 7 days, adding fresh drug and conditioned media (CM) every other day. Cells were trypsinized, replated, and grown under normal growth conditions for 7 days. Colonies were stained using crystal violet and counted using a cell counter. Each experiment was performed in triplicate at least three times. Student's t test was used to determine survival differences between gefitinib-treated cells in the presence or absence of conditioned media.
Figure 4
Figure 4
Met phosphorylation and DNA synthesis in triple-negative breast cancer cells in the presence of gefitinib. Co-culture with hepatocyte growth factor (HGF)-expressing fibroblasts increases Met phosphorylation and DNA synthesis in triple-negative breast cancer cells in the presence of gefitinib. (A) SUM102 cells were labeled with CytoTracker green and were co-cultured with RMF-HGF cells at different ratios. Lysates were analyzed for phosphorylation of Met by immunoblotting. β-actin and total Met protein expression were used as controls. Numbers below the immunoblots represent relative densitometry measurements of the ratio of pMet to total Met. (B) RMF-HGF fibroblasts were plated on coverslips. SUM102 and SUM149 cells were labeled with CytoTracker green and plated on top of the RMF-HGF fibroblasts at the indicated ratios. Then 1.0 μM gefitinib (gef) was added to the cells for 24 hours. SUM102 cells were incubated with BrdU for 18 hours and SUM149 cells were incubated with BrdU for 4 hours. Cells were fixed and stained using anti-BrdU-Alexa-Fluor-595. SUM102 and SUM149 cells labeled green were counted as BrdU-positive or BrdU-negative. Student's t test was used to determine statistical significance between gefitinib-treated cells with or without co-cultures. *SUM102, P = 0.0471; SUM149, P = 0.0028. UT, untreated; 0/10, fibroblast to tumor cell ratio; 10/10, fibroblast to tumor cell ratio.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Korsching E, Jeffrey SS, Meinerz W, Decker T, Boecker W, Buerger H. Basal carcinoma of the breast revisited: an old entity with new interpretations. J Clin Pathol. 2008;14:553–560. doi: 10.1136/jcp.2008.055475. - DOI - PubMed
    1. Baselga J, Albanell J, Ruiz A, Lluch A, Gascon P, Guillem V, Gonzalez S, Sauleda S, Marimon I, Tabernero JM, Koehler MT, Rojo F. Phase II and tumor pharmacodynamic study of gefitinib in patients with advanced breast cancer. J Clin Oncol. 2005;14:5323–5333. doi: 10.1200/JCO.2005.08.326. - DOI - PubMed
    1. Tan AR, Yang X, Hewitt SM, Berman A, Lepper ER, Sparreboom A, Parr AL, Figg WD, Chow C, Steinberg SM, Bacharach SL, Whatley M, Carrasquillo JA, Brahim JS, Ettenberg SA, Lipkowitz S, Swain SM. Evaluation of biologic end points and pharmacokinetics in patients with metastatic breast cancer after treatment with erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor. J Clin Oncol. 2004;14:3080–3090. doi: 10.1200/JCO.2004.08.189. - DOI - PubMed
    1. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J, Haber DA. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;14:2129–2139. doi: 10.1056/NEJMoa040938. - DOI - PubMed
    1. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers WR, Johnson BE, Meyerson M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;14:1497–1500. doi: 10.1126/science.1099314. - DOI - PubMed

Publication types

MeSH terms